Apply the Samples as bands to a suitable HPTLC plate and dry in air. Develop the chromatograms in a saturated chamber, remove the plate from the chamber, and dry. Treat with Derivatization reagent and heat for 5 min at 105°. Immediately examine under visible light and UV 365 nm.

System suitability: Under visible light, Standard solution C exhibits about five purple bands, one near the origin, three around the middle, and one near the solvent front. Under UV 365 nm, Standard solution C exhibits about seven bands with increasing RF: a pale yellow band near the origin, a pale blue band, a brown band, a blue band, a pale white band, and two light brown bands.

Acceptance criteria: Under visible light, the Sample solution exhibits about five bands with increasing RF: one pale purple band, one brown-purple band around RF of 0.3, two purple bands near the middle, and one pale purple band near the solvent front. Under UV 365 nm, the Sample solution exhibits about eight bands below the RF that corresponds to the ergosterol band in Standard solution B. These bands correspond in color and RF to the bands in Standard solution C with increasing RF from the origin: a yellow band, a bright blue band, a brown band, a bright yellow band, a pale yellow band, a brown band, a bright brown band, and a brown band. Some extra pale bands overlap above the brown band.

• B. HPLC

Analysis: Proceed as directed in the Assay for Content of Triterpenoids.

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at the retention times corresponding to the peaks due to antcin A, antcin B, antcin C, antcin H, antcin K, 1,4-dimethoxy-2,3-methylenedioxy-5-methylbenzene, dehydrosulfurenic acid, and dehydroeburicoic acid in Standard solution B.

• C. HPLC​

Analysis: Proceed as directed in the Assay for Content of Antroquinonol.

Acceptance criteria: The chromatogram of the Sample solution exhibits peaks at the retention times corresponding to antroquinonol in Standard solution B.

[Note—Incorporate a factor in the calculation to account for the aliquot taken compared to the volume of the Sample solution.]

Calculate the content of triterpenoids as the sum of the percentages of the different triterpenes.

Separately calculate the percentage of the labeled amount of triterpenoids in the Dry Extract:

Result = (P/L) × 100

P = content of triterpenoids as determined above (%)

L = labeled amount of triterpenoids (%)

Acceptance criteria: 90.0%–110.0% on the dried basis

• Content of Antroquinonol

Solution A: 0.3% Acetic acid in water

Solution B: Methanol

Mobile phase: See Table 3.

Table 3

Time

(min)

Solution A

(%)

Solution B

(%)

0

95

5

10

20

80

20

10

90

35

10

90

40

95

5

Standard solution A: 0.5 mg/mL of USP Antroquinonol RS in methanol

Standard solution B: Sonicate 1.0 mg/mL of USP Antrodia camphorata Fruiting Body Dry Extract RS in methanol for 10 min. Before injection, pass through a PTFE filter of 0.2-μm pore size and discard the first portion of the filtrate.

Sample solution: Transfer about 10 mg of Antrodia camphorata Fruiting Body Dry Extract to a flask and add 10.0 mL of methanol. Sonicate for 30 min and adjust to initial weight with methanol. Before injection, pass through a PTFE filter of 0.2-μm pore size and discard the first portion of the filtrate.

Chromatographic system

(See Chromatography <621> System Suitability.)

Mode: HPLC

Detector: UV 254 nm

Column: 4.6-mm × 25-cm; 5-μm packing L1 (similar to Hypersil C-18)

Flow rate: 1.0 mL/min

Injection volume: 20 μL

System suitability

Samples:Standard solution A and Standard solution B

Suitability requirements

Chromatographic similarity: The chromatogram of Standard solution B is similar to the reference chromatogram provided with the lot of USP Antrodia camphorata Fruiting Body Dry Extract RS being used.

Using the chromatograms of Standard solution A, Standard solution B, and the reference chromatogram provided with the lot of USP Antrodia camphorata Fruiting Body Dry Extract RS being used, identify the retention time of the peak corresponding to antroquinonol.

Calculate the percentage of antroquinonol in the portion of Antrodia camphorata Fruiting Body Dry Extract taken:

• Microbial Enumeration Tests <61>: The total aerobic bacterial count does not exceed 105 cfu/g, the total combined molds and yeasts count does not exceed 103 cfu/g, and the bile-tolerant Gram-negative bacteria does not exceed 103 cfu/g.